Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides

Abstract

Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

Keywords: 5-ethynyl-2′-deoxyuridine; DNA glycosylases; DNA polymerases; DNA repair; click chemistry; metabolic labeling; replication.

Figure 1

Structures of clickable U and C derivatives investigated in this work.

Figure 2

The cleavage of DNA substrates containing C8-AlkU or EtU paired with G (a) or A (b) or in single-stranded DNA (c) by DNA glycosylases. S, substrate; P, cleavage product. The asterisk indicates the ³²P-labeled strand. The structures of the substrates are shown above the gel images; X, modified base. Original images of (a–c) can be found in Supplementary Materials.

Figure 3

Time course of product accumulation under single-turnover conditions during substrate cleavage by SMUG1 (a) and MBD4 (b). Circles and error bars indicate mean ± s.d. of three independent experiments; lines are fits to equation $\left[\mathrm{P}\right]=A\left(1-e^{-k_{2\mathrm{a}\mathrm{p}\mathrm{p}}t}\right)$ (see Section 2.3). Black symbols, EtU:G substrate; red, C8-AlkU:G; green, U:G; cyan, T:G.

Figure 4

Primer extension by KF (a) and RBpol (b) across EtU, C8-AlkU and C8-AlkC. The template modification and dNTPs are indicated under the gel images. N, an equimolar mixture of all four dNTPs. P, primer. The structures of the substrates and the nature of the polymerase are shown above the gel images; X, modified base; the asterisk indicates the fluorescein-labeled primer. Original images of (a,b) can be found in Supplementary Materials.

Figure 5

Primer extension by POLβ on the primer–template and gapped substrates across EtU (a), C8-AlkU (b) and C8-AlkC (c). The template modification and dNTPs are indicated under the gel images. N, an equimolar mixture of all four dNTPs. P, primer. The structures of the substrates and the nature of the polymerase are shown above the gel images; X, modified base; the asterisk indicates the fluorescein-labeled primer. PT, primer–template substrate; gap, 1 nt gap substrate. Original images of (a–c) can be found in Supplementary Materials.

Figure 6

Primer extension by POLλ (a) and POLκ (b) across EtU, C8-AlkU and C8-AlkC. The template modification and dNTPs are indicated under the gel images. N, an equimolar mixture of all four dNTPs. P, primer. The structures of the substrates and the nature of the polymerase are shown above the gel images; X, modified base; the asterisk indicates the fluorescein-labeled primer. Original images of (a,b) can be found in Supplementary Materials.

Figure 7

Transcriptional mutagenesis and repair of EtU and C8-AlkU in HeLa cells. Relative EGFP expression normalized for the fluorescence of the control G-construct is presented (n = 3, mean ± SD shown). p < 0.05 (*); p < 0.005 (***); p < 0.001 (****), two-tailed Student’s t-test with Bonferroni correction.